Antibody-Mediated Immobilization of Virions in Mucus.

Antibodies have been shown to hinder the movement of herpes simplex virus virions in cervicovaginal mucus, as well as other viruses in other mucus secretions. However, it has not been possible to directly observe the mechanisms underlying this phenomenon, so the nature of virion-antibody-mucin interactions remain poorly understood. In this work, we analyzed thousands of virion traces from single particle tracking experiments to explicate how antibodies must cooperate to immobilize virions for relatively long time periods. First, using a clustering analysis, we observed a clear separation between two classes of virion behavior: freely diffusing and immobilized. While the proportion of freely diffusing virions decreased with antibody concentration, the magnitude of their diffusivity did not, implying an all-or-nothing dichotomy in the pathwise effect of the antibodies. Proceeding under the assumption that all binding events are reversible, we used a novel switch-point detection method to conclude that there are very few, if any, state switches on the experimental timescale of 20 s. To understand this slow state switching, we analyzed a recently proposed continuous-time Markov chain model for binding kinetics and virion movement. Model analysis implied that virion immobilization requires cooperation by multiple antibodies that are simultaneously bound to the virion and mucin matrix and that there is an entanglement phenomenon that accelerates antibody-mucin binding when a virion is immobilized. In addition to developing a widely applicable framework for analyzing multistate particle behavior, this work substantially enhances our mechanistic understanding of how antibodies can reinforce a mucus barrier against passive invasive species.

Case Reports: Prolonged Detection of Zika Virus RNA in Vaginal and Endocervical Samples from a Brazilian Woman, 2018.

Zika virus (ZIKV) infection is an emerging public health problem, associated with increased risk for Guillain-Barré syndrome and adverse fetal outcomes, including congenital microcephaly. Zika virus sexual transmission is known, but detection of the virus in different parts of the female reproductive tract is not well established. In this case report, we describe prolonged detection of ZIKV RNA in the vaginal secretion and endocervical mucosa from a Brazilian woman convalescent to ZIKV infection. A viral load of 2 × 102 copies/mL was detected up to 31 days after symptom onset in both samples. Other biological fluids, including whole blood, plasma, serum, urine, and saliva samples, were negative for ZIKV RNA. These findings advance the understanding of ZIKV infection and provide data for additional testing strategies.

Herpes simplex virus-binding IgG traps HSV in human cervicovaginal mucus across the menstrual cycle and diverse vaginal microbial composition.

IgG possesses an important yet little recognized effector function in mucus. IgG bound to viral surface can immobilize otherwise readily diffusive viruses to the mucin matrix, excluding them from contacting target cells and facilitating their elimination by natural mucus clearance mechanisms. Cervicovaginal mucus (CVM) is populated by a microbial community, and its viscoelastic and barrier properties can vary substantially not only across the menstrual cycle, but also in women with distinct microbiota. How these variations impact the "muco-trapping" effector function of IgGs remains poorly understood. Here we obtained multiple fresh, undiluted CVM specimens (n = 82 unique specimens) from six women over time, and employed high-resolution multiple particle tracking to quantify the mobility of fluorescent Herpes Simplex Viruses (HSV-1) in CVM treated with different HSV-1-binding IgG. The IgG trapping potency was then correlated to the menstrual cycle, and the vaginal microbial composition was determined by 16 s rRNA. In the specimens studied, both polyclonal and monoclonal HSV-1-binding IgG appeared to consistently and effectively trap HSV-1 in CVM obtained at different times of the menstrual cycle and containing a diverse spectrum of commensals, including G. vaginalis-dominant microbiota. Our findings underscore the potential broad utility of this "muco-trapping" effector function of IgG to reinforce the vaginal mucosal defense, and motivates further investigation of passive immunization of the vagina as a strategy to protect against vaginally transmitted infections.

Imaging and tracking HIV viruses in human cervical mucus.

We describe a systematic approach to image, track, and quantify the movements of HIV viruses embedded in human cervical mucus. The underlying motivation for this study is that, in HIV-infected adults, women account for more than half of all new cases and most of these women acquire the infection through heterosexual contact. The endocervix is believed to be a susceptible site for HIV entry. Cervical mucus, which coats the endocervix, should play a protective role against the viruses. Thus, we developed a methodology to apply time-resolved confocal microscopy to examine the motion of HIV viruses that were added to samples of untreated cervical mucus. From the images, we identified the viruses, tracked them over time, and calculated changes of the statistical mean-squared displacement (MSD) of each virus. Approximately half of tracked viruses appear constrained while the others show mobility with MSDs that are proportional to ??+?2?2, over time range ?, depicting a combination of anomalous diffusion (0

Thermosensitive and mucoadhesive pluronic-hydroxypropylmethylcellulose hydrogel containing the mini-CD4 M48U1 is a promising efficient barrier against HIV diffusion through macaque cervicovaginal mucus.

To be efficient, vaginal microbicide hydrogels should form a barrier against viral infections and prevent virus spreading through mucus. Multiple particle tracking was used to quantify the mobility of 170-nm fluorescently labeled COOH-modified polystyrene particles (COOH-PS) into thermosensitive hydrogels composed of amphiphilic triblock copolymers with block compositions EOn-POm-EOn (where EO refers to ethylene oxide and PO to propylene oxide) containing mucoadhesive hydroxypropylmethylcellulose (HPMC). COOH-PS were used to mimic the size and the surface charge of HIV-1. Analysis of COOH-PS trajectories showed that particle mobility was decreased by Pluronic hydrogels in comparison with cynomolgus macaque cervicovaginal mucus and hydroxyethylcellulose hydrogel (HEC; 1.5% by weight [wt%]) used as negative controls. Formulation of the peptide mini-CD4 M48U1 used as an anti-HIV-1 molecule into a mixture of Pluronic F127 (20 wt%) and HPMC (1 wt%) did not affect its anti-HIV-1 activity in comparison with HEC hydrogel. The 50% inhibitory concentration (IC50) was 0.53 µg/ml (0.17 µM) for M48U1-HEC and 0.58 µg/ml (0.19 µM) for M48U1-F127-HPMC. The present work suggests that hydrogels composed of F127-HPMC (20/1 wt%, respectively) can be used to create an efficient barrier against particle diffusion in comparison to conventional HEC hydrogels.

Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions.

Cervical cancer induced by human papillomavirus (HPV) is the fourth highest mortality causing cancer in women despite the use of prophylactic vaccines. E6 targeting represents an attractive strategy to treat this cancer. Indeed, oncoprotein E6 is produced by keratinocytes infected by HPV and is partially responsible for carcinogenesis. E6 interferes with the apoptosis process in stressed cells by degradation of p53 tumor suppressor gene. Our strategy consists in using E6 siRNA complexed with pegylated lipoplexes. The addition of hydrophilic polymer around the nanoparticles is crucial to use them by vaginal application on account of cervicovaginal mucus. Physicochemical characteristics were evaluated and in vitro assays were performed to evaluate transfection potential, E6 mRNA extinction and p53 re-expression. Cationic liposomes DOTAP/Cholesterol/DOPE 1/0.75/0.5 (N/P 2.5) with or without 50% DSPE-PEG2000 and associated with siE6 have demonstrated good physicochemical characteristics in terms of complexation, size, surface charge and stability. Both lipoplexes have been tested on CaSki cell line (HPV 16+) with 50 nM and 100 nM of siE6. Lipoplexes formulations induce 30-40% of E6 mRNA extinction and induce the re-expression of p53. In conclusion, pegylated anti-E6 lipoplexes have demonstrated their efficiency to cross the cellular membrane and to release siRNA into the cytoplasm confirmed by final p53 protein production.

Pretreatment of human cervicovaginal mucus with pluronic F127 enhances nanoparticle penetration without compromising mucus barrier properties to herpes simplex virus.

Mucosal drug delivery nanotechnologies are limited by the mucus barrier that protects nearly all epithelial surfaces not covered with skin. Most polymeric nanoparticles, including polystyrene nanoparticles (PS), strongly adhere to mucus, thereby limiting penetration and facilitating rapid clearance from the body. Here, we demonstrate that PS rapidly penetrate human cervicovaginal mucus (CVM), if the CVM has been pretreated with sufficient concentrations of Pluronic F127. Importantly, the diffusion rate of large polyethylene glycol (PEG)-coated, nonmucoadhesive nanoparticles (PS-PEG) did not change in F127-pretreated CVM, implying that F127 did not significantly alter the native pore structure of CVM. Additionally, herpes simplex virus type 1 (HSV-1) remains adherent in F127-pretreated CVM, indicating that the presence of F127 did not reduce adhesive interactions between CVM and the virions. In contrast to treatment with a surfactant that has been approved for vaginal use as a spermicide (nonoxynol-9 or N9), there was no increase in inflammatory cytokine release in the vaginal tract of mice after daily application of 1% F127 for 1 week. Pluronic F127 pretreatment holds potential as a method to safely improve the distribution, retention, and efficacy of nanoparticle formulations without compromising CVM barrier properties to pathogens.

A prospective cohort study of the effect of depot medroxyprogesterone acetate on detection of plasma and cervical HIV-1 in women initiating and continuing antiretroviral therapy.

Depot medroxyprogesterone acetate (DMPA) use among HIV-1-infected women may increase transmission by increasing plasma and genital HIV-1 RNA shedding. We investigated associations between DMPA use and HIV-1 RNA in plasma and cervical secretions. One hundred two women initiated antiretroviral therapy, contributing 925 follow-up visits over a median of 34 months. Compared with visits with no hormonal contraception exposure, DMPA exposure did not increase detection of plasma (adjusted odds ratio: 0.81, 95% confidence interval: 0.47 to 1.39) or cervical HIV-1 RNA (adjusted odds ratio: 1.41, 95% confidence interval: 0.54 to 3.67). Our results suggest that DMPA is unlikely to increase infectivity in HIV-positive women who are adherent to effective antiretroviral therapy.

Frequent detection of cytomegalovirus and Epstein-Barr virus in cervical secretions from healthy young women.

OBJECTIVE: To investigate asymptomatic shedding from the uterine cervix of five human herpes viruses: cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) type 1 and type 2 and varicella zoster virus (VZV), in young women. DESIGN: A descriptive study. SETTING: Sahlgrenska University Hospital. POPULATION: Three hundred and five young, healthy Swedish women. METHODS: Cervical specimens were analyzed for the presence of viral DNA with a quantitative real-time PCR assay. MAIN OUTCOME MEASURES: Detection of viral DNA. RESULTS: Viral DNA was detected in 66 (21.6%) of the cervical samples. The most common findings were CMV DNA, detected in 35 (11.5%), and EBV DNA, found in 32 (10.5%) of the women. HSV-1 DNA was detected in 5 (1.7%) and HSV-2 DNA in 4 (1.4%), but VZV DNA was not found. The estimated DNA level for the detected viruses was similar with a mean DNA quantity of 2.6 log genome equivalents (Geq)/mL for CMV (range 1.7-4.3), 2.5 log Geq/mL for EBV (range 1.7-4.7), 2.4 log Geq/mL for HSV-1 (range 1.7-3.5) and 2.6 log Geq/mL for HSV-2 (range 1.7-4.1). The simultaneous presence of DNA from two or more herpes viruses was detected in eight specimens. CONCLUSIONS: Asymptomatic shedding of CMV and EBV from the uterine cervix was found in one-fifth of young women. In four of the cervical samples; two with EBV, one with CMV, one with HSV-2, high amounts of viral DNA (>4 log Geq/mL) were detected suggesting a greater risk of transmitting the virus perinatally or sexually.

Human cervicovaginal mucus contains an activity that hinders HIV-1 movement.

Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

[Cytokine status of cervical mucus in women with transitory and persistent course of papillomavirus infection].

AIM: Study the levels of cytokines in cervical mucus of women with transitory and persistent course of papillomavirus infection (PVI). MATERIALS AND METHODS: Material from 122 women was studied: 32 patients in control group, 68 patients with transitory course of PVI and 52 women with persistent course of PVI. The presence or absence of high risk HPV DNA was confirmed by real time PCR. High risk HPV DNA was determined in scrapes of epithelial cells of cervical canal. Quantitative determination of cytokines (IFN-alpha, IFN-gamma, IL-1beta, receptor antagonist IL-1, IL-2, IL-4, IL-8 and IL-10, TNFalpha in cervical mucus was performed by enzyme immunoassay. RESULTS: Transitory course of PVI was characterized by an increase of IFN-alpha and IFN-gamma, persistent course of PIV--by a decrease of IFN-alpha, IL-2 level was increased in both groups of patients with PVI. CONCLUSION: The outcome of PVI is controlled by interferon component of the immunity. Because of this during observation of patients with PVI the determination of IFN-alpha, IFN-gamma content in cervical mucus is an appropriate diagnostic procedure in clinical practice for the prognosis of risk of development of high risk PIV persistency.

Presence of DNA of adeno-associated virus in subfertile couples, but no association with fertility factors.

BACKGROUND: Based on previous reports suggesting a role of adeno-associated virus (AAV) in miscarriage, the prevalence of AAV DNA in genital tracts of male and female partners of subfertile couples was determined to assess a potential association of AAV infection with clinically relevant parameters of male and female fertility. METHODS: A prospective study was performed in the outpatient infertility clinic of a university-based hospital. Semen samples and endocervical material obtained from 146 male and 134 female partners of asymptomatic subfertile couples were analyzed for the presence of AAV DNA (using nested PCR). Patients' medical histories and details of clinical examinations were recorded. Semen quality, including sperm functional capacity and the presence of antisperm antibodies (ASA) and seminal white blood cells (WBC), was assessed in aliquots of the same ejaculate. Detailed examinations of the cervical factor and other variables of female subfertility were performed. Both partners were screened for bacterial infection. RESULTS: The presence of AAV DNA in semen was not significantly related to semen quality, including sperm functional capacity or local ASA, nor was it coupled to the presence of AAV in the endocervical material of female partners. The presence of AAV DNA was not associated with the presence of other micro-organisms of the lower genital tract or with seminal WBC in men. AAV DNA in endocervical material was not related to a reduced quality of cervical mucus or to other female infertility factors. CONCLUSIONS: The presence of AAV DNA in semen samples or endocervical swabs showed no significant association with clinically relevant infertility factors. However, longitudinal studies may clarify previous suggestions of an influence of AAV infection on early pregnancy problems.

Shedding dynamics of Epstein-Barr virus: A type 1 carcinogen.

Epstein Barr virus (EBV) is one of the ubiquitous viral carcinogens found in humans and successfully infects more than 90% of the world population. The spectrum of EBV-related pathology ranges from asymptomatic primary infection to grave B- and T-cell malignancies. EBV triggers lymphoproliferative disorders after allogeneic stem cell transplantation, which contributes to higher mortality rates. Although the transmission of EBV primarily occurs from an infected host to a naive host through viral shedding from the oropharynx, increasing evidence points to considerable amount of shedding in other anatomical sites such as cervix, anal mucosa, breast milk and respiratory tract. It is impossible to eradicate the prevalence of EBV-related malignancies and other pathologies without preventing viral shedding. However, a detail analysis of the multifaceted nature of EBV shedding is not available in the literature. Thus, this review focuses on elucidating the key elements of the shedding dynamics of this carcinogenic virus.

Prevalence of human papillomavirus genotypes, and mucosal IgA anti-viral responses in women with cervical ectopy.

BACKGROUND: Data on the prevalence of different human papillomavirus (HPV) genotypes and the associated mucosal immune response in women with cervical ectopy are scarce. OBJECTIVE: To assess the prevalence of different HPV genotypes and the mucosal anti-viral immune response in cervical ectopy. STUDY DESIGN: Detection and typing of HPV DNA was determined in 141 women with cervical ectopy, 272 cytologically normal controls and 98 low-grade squamous intraepithelial lesions (LSIL) by PCR and direct sequencing. Mucosal IgA antibodies to HPV16 and HPV18 were evaluated in cervical mucus by ELISA. RESULTS: The prevalence of HPV in cervical ectopy was higher (73.7%) than that observed in control samples (30.5% in endocervix, and 1.8% in exocervix), but similar to the prevalence in LSIL (62.2%). Typing showed that the overall distribution frequency concerned 14 different genotypes, with HPV18 being the most prevalent in cervical ectopy (53.9%), whereas HPV16 predominated in LSIL (38.7%). High-risk HPV genotypes were 2.2 times more frequent in cervical ectopy than in the normal endocervix (p<0.0001). HPV infection in cervical ectopy patients was accompanied by a mucosal IgA-antibody response. Antibody reactivity to HPV18 was significantly higher than the response to HPV16. CONCLUSION: Cervical ectopy is a risk factor for infection with high-risk HPV genotypes, in particular HPV18. Our results emphasize the need of further studies to clarify the oncogenic potential of this virus in cervical ectopy.

Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses.

The mechanisms by which mucus helps prevent viruses from infecting mucosal surfaces are not well understood. We engineered non-mucoadhesive nanoparticles of various sizes and used them as probes to determine the spacing between mucin fibers (pore sizes) in fresh undiluted human cervicovaginal mucus (CVM) obtained from volunteers with healthy vaginal microflora. We found that most pores in CVM have diameters significantly larger than human viruses (average pore size 340 +/- 70 nm; range approximately 50-1800 nm). This mesh structure is substantially more open than the 15-100-nm spacing expected assuming mucus consists primarily of a random array of individual mucin fibers. Addition of a nonionic detergent to CVM caused the average pore size to decrease to 130 +/- 50 nm. This suggests hydrophobic interactions between lipid-coated "naked" protein regions on mucins normally cause mucin fibers to self-condense and/or bundle with other fibers, creating mucin "cables" at least three times thicker than individual mucin fibers. Although the native mesh structure is not tight enough to trap most viruses, we found that herpes simplex virus (approximately 180 nm) was strongly trapped in CVM, moving at least 8,000-fold slower than non-mucoadhesive 200-nm nanoparticles. This work provides an accurate measurement of the pore structure of fresh, hydrated ex vivo CVM and demonstrates that mucoadhesion, rather than steric obstruction, may be a critical protective mechanism against a major sexually transmitted virus and perhaps other viruses.

Movements of HIV-virions in human cervical mucus.

Time-resolved confocal microscopy and fluorescence correlation spectroscopy were used to examine the movements of fluorescently labeled HIV-virions (approximately 100 nm) added to samples of human cervical mucus. Particle-tracking analysis indicates that the motion of most virions is decreased 200-fold compared to that in aqueous solution and is not driven by typical diffusion. Rather, the time-dependence of their ensemble-averaged mean-square displacements is proportional to tau(alpha) + v(2)tau(2), describing a combination of anomalous diffusion (alpha approximately 0.3) and flow-like behavior, with tau being the lag time. We attribute the flow-like behavior to slowly relaxing mucus matrix that follows mechanical perturbations such as stretching and twisting of the sample. Further analysis of the tracks and displacements of individual virions indicates differences in the local movements among the virions, including constrained motion and infrequent jumps, perhaps due to abrupt changes in matrix structure. Changes in the microenvironments due to slow structural changes may facilitate movement of the virions, allowing them to reach the epithelial layer.

Epidemiology of HPV in HIV-positive and HIV-negative fertile women in Cameroon, West Africa.

BACKGROUND: HPV types vary by country and HIV status. There are no data on the prevalent HPV genotypes from Cameroon. METHODS: We conducted a cross-sectional, observational study on 65 Cameroonian women. Samples were sent for HPV genotyping and Thin Prep analyses. RESULTS: 41 out of 61 samples tested (67.2%) had HPV subtypes detected. The most common high risk types encountered were: 45 (24.6%) and 58 (21.5%). HIV-positive women were more likely to test positive for any HPV (P = .014), have more than one HPV subtype (P = .003), and to test positive for the high risk subtypes (P = .007). Of those with high risk HPV, HIV-positive women were more likely to have Thin Prep abnormalities than HIV-negative women (P = .013). CONCLUSIONS: Oncogenic HPV subtypes 45 and 58 were more prevalent than those subtypes carried in the quadrivalent vaccine. Further studies are needed to assess whether the current vaccine will be effective in this region.

HIV type 1 zidovudine (ZDV) resistance in blood and uterine cervical secretions of pregnant women.

To determine if HIV-1 resistance testing of the blood predicts mutants in the genital secretions of ZDV-treated pregnant women, ZDV resistance mutations were assessed by an oligonucleotide ligation assay. ZDV resistance was detected in viral sequences from blood (16/48; 33%) and cervical secretions (6/24; 25%) collected during 57 pregnancies. The genotype of 11/69 (16%) resistance codons was discordant between blood and cervical virus of pregnant women near term. However, in only 1 (1.8%) of 57 pregnancies evaluated was resistant virus limited to the cervical secretions. ZDV-resistant virus is rarely limited to the female genital tract.

Enterovirus RNA shedding in the genital tract of childbearing-aged women living in Central Africa.

Enteroviruses (EVs) (Picornaviridae) in the female genital tract may constitute possible sources of antenatal or perinatal infection. The presence of EV genomes in the acellular part of cervicovaginal lavages of 119 non-pregnant childbearing-aged African women was determined using a semiquantitative RT-PCR and hybridization detection assay. EV-specific cervicovaginal IgA and IgG antibodies were also detected by immunocapture ELISA assays. Of 119 CVS samples tested, only 10 (8%) were positive for the detection of EV RNA, demonstrating an genital shedding of EVs in African woman. EV-RNA positivity was not associated with the HIV serostatus or with the presence of semen traces in female genital secretion. The microwell hybridization assay of EV amplified RT-PCR products indicated the presence of low levels of EV genomes, ranging from 50 to 100 RNA copies per ml of genital fluids. EV-specific cervicovaginal IgA or IgG antibodies were detected only in two hemoglobin-positive cervicovaginal secretions samples from women without genital EVs. The lack of EV specific IgA or IgG antibody secretion by the cervicovaginal mucosa supported the hypothesis of genital shedding of EVs without ongoing viral replication in the female genital tract. In conclusion, the findings demonstrated the presence of EV genomes in nearly 10% of childbearing-aged women living in Central Africa, and provided the basis of possible antenatal or perinatal transmission of EV from mother-to-child.

HIV binding, penetration, and primary infection in human cervicovaginal tissue.

We have developed human cervicovaginal organ culture systems to examine the initiating events in HIV transmission after exposure to various sources of HIV infectivity, including semen. Newly infected cells were detected in the cervical submucosa 3-4 days after exposure to a primary HIV isolate. At earlier times, extensive and stable binding occurred when cervical surfaces were exposed to virions or seminal cells. Cervical mucus provided some protection for the endocervical surface, by physically trapping virions and seminal cells. Confocal microscopy combined with 3D surface reconstruction revealed that virions could both bind to the external surface of the cervical epithelium and actually penetrate beneath the epithelial surface. In quantitative assays, pretreatment with a blocking antibody directed against beta1 integrin reduced HIV virion binding. Collectively, these results highlight a continuum of complex interactions that occurs when natural sources of HIV infectivity are deposited onto mucosal surfaces in the female reproductive tract.